A typical disease resulting from abnormally elevated migration ability and invasion ability of cells is cancers. Metastasis is mentioned as one of the largest threat of cancers. Metastatic cancer cells migrate from a primary tumor through a blood vessel by cell migration, invade within the blood vessel, and then metastasize to other tissues through the blood stream. PAR1 (protease activated receptor 1) is a seven-transmembrane receptor which activates the migration ability and invasion ability of cancer cells, and is involved in the metastasis of a great variety of cancers (breast cancer, lung cancer, pancreas cancer, prostate cancer, etc.). It is known that, in the breast cancer in particular, PAR1 is expressed in most of cultured cancer cell lines having a metastatic ability, and that PAR1 is activated when the N-terminal extracellular domain (between R4 1 and S4 2) of the PAR1 is cleaved by a protease inherent to cancers (MMP1: matrix metalloprotease 1). The stimulation of PAR1 activation activates G protein coupled to an intracellular domain of the PAR1. It is thought that an intracellular Ca2+ concentration is locally elevated as a result of the activation of the G protein, and the cell migration ability and cell invasion ability are enhanced by the Ca signal (see Non-Patent Document 1).
PAR1 was originally discovered as a thrombin-dependent receptor (thrombin receptor) necessary for activating platelets. Thrombin recognizes and cleaves the same site in the PAR1 as in the case of the MMP1. Also, activation of the thrombin receptor in platelets activates the Ca2+ signal in a manner similar to that in the case of cancer cells. Currently, some monoclonal antibodies to an N-terminal cell domain of the PAR1 are marketed, which antibodies inhibit the activation of platelets. However, there is no report as to the relationship between these antibodies and the migration and invasion of cancer cells.
Even more, there is no report describing that the migration and invasion of cancer cells were inhibited by a PAR1 antibody, or that a PAR1 antibody was used for a cancer treatment.
Also, a substance named Pepducin was prepared by synthesizing a peptide corresponding to a G protein binding site of the PAR1 and modifying the peptide so as to have membrane permeability (see Non-Patent Document 2). Covic et al. succeeded in inhibiting the activity of the PAR1 by utilizing Pepducin as a G protein antagonist (inhibiting effect of Pepducin is 3 to 4 μM). On the other hand, a method applying an antibody as an anti-cancer drug is expected to show not only an effect inhibiting an activity of a target molecule but also an effect exerting an antibody-dependent cell impairing activity by immune system cells. In addition, a technique for preparing antibodies having a high affinity by genetic engineering has been established, and the safety of antibodies to a living body has been ensured. For the reasons as described above, there is a very large merit in applying antibodies as an anti-cancer drug. Accordingly, it is thought that the application of a PAR1 antibody as an anti-cancer drug has a possibility that the antibody becomes a more effective anti-cancer drug than Pepducin.
An antibody in which the epitope is a sequence from fifty-second Tyr to fifty-sixth Trp in the PAR1 was prepared by a phage display method, which antibody inhibits an activity of thrombin cleaving the PAR1. With respect to the inhibition of the cleaving activity of the PAR1 by the antibody (binding to PAR1), in particular, it is thought that the inclusion of fifty-sixth Trp in the epitope is important (see Patent Document 1). Currently, there is no report describing that the migration and invasion of cancer cells were inhibited by a PAR1 antibody, or that other peptide sequence was found as an epitope for the PAR1 antibody effective for an inhibitory effect.
Patent Document 1: JP-2002-10784 A Non-Patent Document 1: Boire A, Covic L, Agarwal A, Jacques S, Sherifi S, Kuliopulos A, PAR1 is a matrix matalloprotease-1 receptor that promotes invasion and tumorigenesis of breast cancer cells. Cell 120: 303-313 (2005)
Non-Patent Document 2: Covic L, Misra M, Bader Singh C, Kuliopulus A, Pepducin-based intervention of thrombin-receptor signaling and systematic platelet activation. Nat. Med. 8; 1161-1165 (2002)